Association of CTLA-4 and IL-4 polymorphisms in viral induced liver cancer

Background Hepatocellular carcinoma (HCC) is one of the most prevalent types of cancer and is responsible for close to one million annual deaths globally. In Pakistan, HCC accounts for 10.7% of cancer incidence. Prior studies indicated an association between interleukin 4 (IL-4) and cytotoxic T lymphocyte protein 4 (CTLA-4) gene polymorphisms in many types of cancers, including HCC that are either hepatitis B virus (HBV)- or hepatitis C Virus (HCV)-induced. The association of IL-4 and CTLA-4 genetic polymorphisms with HCV-induced HCC is not yet determined in the Pakistani population. Therefore, this research is designed to investigate the implication of IL-4 and CTLA-4 gene polymorphisms by determining the association of IL-4 -590 C/T (rs2243250) and CTLA-4 + 49 A/G (rs231775) with HCC in Pakistan. Methods Different bioinformatics tools were employed to determine the pathogenicity of these polymorphisms. Samples were collected from HCV-induced HCC patients, followed by DNA extraction and ARMS-PCR analysis. Results The SNP analysis results indicated a positive association of IL-4 -590C/T and CTLA-4 + 49A/G gene polymorphisms with HCV-induced HCC in Pakistan. The CTLA-4 polymorphism might enhance therapeutic efficiency of HCC chemotherapy medicines. The IL-4 polymorphism might introduce new transcription factor binding site in IL-4 promoter region. Conclusion This study delineated risk factor alleles in CTLA-4 and IL-4 genes associated with HCV-mediated HCC among Pakistani patients that may have application to serve as genetic markers for pre- and early diagnosis and prognosis of HCC in HCV patients.


Background
Hepatocellular carcinoma (HCC) is a multifactorial primary liver malignancy caused by excessive alcohol intake, obesity and viral infections [1]. Globally, hepatitis B and C virus infections are a major cause of HCC. Chronic HBV and HCV infections have been responsible for 44% and 22% of HCC cases, respectively [2]. HCC is responsible for almost 830,180 deaths around the globe [3]. In Pakistan, about 70% of HCC is imputed to HCV, whereas HBV is the major etiological contributor of HCC disease in Asian and Pacific countries [4].
HCV promotes generation of cytokines which contribute to HCC progression. Cancer-associated genetic polymorphisms have been identified in cytokines that might facilitate their targeting for anti-cancer therapies [5]. Cytokine IL-4 is involved in regulation of various important biological functions including B-cell mitogenesis, class switching to IgE, cell homeostasis, and tissue repair [6]. During T-cell signalling, IL-4 promotes T-cell differentiation. In cancer, IL-4 functions in the tumor microenvironment to mediate pro-tumor activity by activating tumor-associated or myeloid-derived suppressor cell (MDSC) associated macrophages [7]. Compared to IL-4, CTLA-4CTLA-4 acts as a negative regulator of T-cell signalling. It is present on T-cells and binds B7-1 (CD-80) and B7-2 ligands (CD-86). It induces changes that directly stop TCR immune synapse and block CD28 signaling, thus downregulating interaction of T-cells with antigen-presenting cells [8]. Antibodies that target CTLA-4CTLA-4 are used clinically as anti-cancer immunotherapeutic agents [9].
Genetic variants of IL-4 and CTLA-4 are reported to have association with several cancers including HCC, colorectal cancer, and head and neck cancer [10][11][12][13][14]. A recent publication indicated an IL-4IL-4 variant (rs2243250) with relation to HCV-induced HCC in an Egyptian population [15]. Previously, its association was delineated in a Chinese population [16]. Similarly, association between CTLA-4 rs231775 and HCV-induced HCC was found in the Chinese Han population [17] and an Egyptian population [18]. Previous studies have delineated the contribution of pathogenic single nucleotide polymorphisms (SNPs) on clinical outcomes in cancer patients. The presence of certain SNPs boosts the efficiency of some treatment drug's efficacy, leading to increased patient survival rate. For example, the genetic variant rs9582036 was identified in the VEGFR1 receptor that increased patient survival after treatment with bevacizumab [19]. Likewise, HCC patients having the KDR gene rs1870377 AA genotype were reported to show better response to first-line therapy sorafenib [19].
In cancer, CTLA-4 mediated modulation of IL-4IL-4 is responsible for immune dysregulation [20]. During HBV infection, up-regulated expression of CTLA-4 results in the increased activity of IL-4 that functions to generate anti-infection response [21]. During HIV infection, CTLA-4 and IL-4 polymorphism influenced the treatment response in patients [22]. Similarly, CTLA-4 and IL-4 polymorphism is also reported to induce HCC progression [23]. The association of genetic polymorphisms of IL-4 and CTLA-4 with HCV-mediated HCC is not investigated in the Pakistani population. The objectives of the current research include the prediction of pathogenicity of IL-4 -590 C/T (rs2243250) and CTLA-4 + 49 A/G (rs231775) and the study of association between IL-4 -590 C/T (rs2243250) and CTLA-4 + 49 A/G (rs231775) polymorphisms with HCV-induced HCC. The IL-4 polymorphism lies in the promoter region; therefore, the potential influence of IL-4 -590 C/T polymorphism on the regulation of IL-4 expression was also estimated.
Structural change in CTLA-4 protein due to the polymorphism was predicted and investigation for CTLA-4 structural changes on the therapeutic efficiency of HCC first and second line therapeutics was carried out. This study determined putative pre-diagnostic genetic marker for HCV-induced HCC and provided insight on functional impact of IL-4 and CTLA-4 genetic variants in HCV-mediated HCC.

SNP Analysis
Pathogenicity of IL-4 SNP (Variant ID: rs2243250) and CTLA-4 SNP (ID: rs231775) was predicted using the following six tools: SIFT, PolyPhen2.0, REVEL, Met-aLR, MutationAssessor and CADD [26]. SIFT and Poly-Phen2.0 predicts protein function changes based on homology in the primary sequence and physiochemical relationship between neighboring amino acids. REVEL integrates prediction scores of SIFT, PolyPhen, Muta-tionAssessor, MetaLR, REVEL, and CADD to make a pathogenicity prediction for a variant. MetaLR uses allele frequency and logistic regression integrating variant deleteriousness scores to predict impact of missense variants. MutationAssessor employs evolutionary conservation knowledge for predicting functionality alteration of the protein on the basis of homology. CADD is used for the prediction of SNP variants deleteriousness via functional or evolutionary conservation data. Regulome DB was used to determine the coordinates, rank, and score of the SNPs [27].

Transcription regulation prediction
Alibaba 2.0 was employed to predict the alteration in transcription factor binding sites (www. gene-regul ation. com). HOPE project analysis was used to determine protein structural alteration and predict resultant pathogenicity (www. cmbi. umcn. nl/ hope/).

In situ mutagenesis
Protein tertiary structure for CTLA-4 was retrieved from Alphafold database (AF-P16410-F1). Mutation in wildtype CTLA-4 structure was introduced through the mutagenesis tool of PyMol v4.0.2. Both mutated and wildtype structures of CTLA-4 were superimposed to highlight differences.

In vitro method Study design and patient selection
A retrospective case-control study was conducted in which patients were recruited from the Combined Military Hospital Rawalpindi, Pakistan. The sample size (N) was 429, out of which 213 were HCC patients and 216 were control patients. The inclusion criterion was set as HCV-infected HCC patients and all other HCC patients were excluded. The confirmation of HCV infection in patients was achieved by quantifying the viral load through quantitative-reverse transcriptase polymerase chain reaction (qRT-PCR). Isolation of HCV RNA from the patients' plasma was performed through Instant virus RNA kit CE IVD ªAnalytik Jena. TaqMan probes were employed for quantifying the HCV in iQ5 real-time PCR detection system ªBio-Rad Laboratories. Healthy subjects were used as control. Sample size was estimated through the procedure explained by Suresh and Chandrashekara (2015) [29] and validated using the G*Power Software version 3.1.9.2 for Windows. Sample collection was performed after written consent was obtained from the subjects. The study was approved by the Institutional Review Board committee, National University of Sciences and Technology, Islamabad, Pakistan (IRB No. 04-2019-03/06). The study was performed in accordance to the principles of the Declaration of Helsinki [30].

Design of primers
Primer1 (http:// prime r1. soton. ac. uk/ prime r1. html) was used for designing the allele-specific primer sets for IL4 -590 C/T (rs2243250) and CTLA4 + 49 A/G genes. For CTLA4, forward and reverse primer sequences used as internal controls were CAC AAG GCT CAG CTG AAC CTG GAT G (for Allele A) and ACA GGA GAG TGC AGG GCC AGG TCC TAGT (for Allele B), respectively. Forward and reverse outer primers were GTG GGT TCA AAC ACA TTT CAA AGC TTC AGG and TCC ATC TTC ATG CTC CAA AAG TCT CACTC, respectively. For IL4, forward and reverse primer sequences used as internal controls were TCA CGG ATT TCT GTT GTG TTTC and GCC TCC CAA CCA TTC CCT TA, respectively. While ACA CTA AAC TTG GGA GAA CAT TGT C for detection of C allele and ACA CTA AAC TTG GGA GAA CAT TGT T for T allele detection for used.

DNA extraction
Total DNA was isolated from blood using standardized phenol-chloroform protocol [31].

Tetra amplification refractory mutation system polymerase chain reaction (Tetra ARMS-PCR)
Tetra ARMS-PCR was used for identification of CTLA-4 + 49 A/G and IL-4 -590 C/T polymorphism in genes. Reaction mixture (total volume 20 µl) was prepared by adding 5X buffer solution (4μL), 2 mM dNTPs (2μL), Taq polymerase (0.5 U), primer (10 pmol each), distilled water (10.9 μL) and DNA template (1 μL). The conditions used in PCR were: Denaturation (95˚C for 4 min), 25 cycles of denaturation (95 °C for 45 s), annealing (58 °C for 45 s), and extension (72 °C for 45 s), and a final extension (72 °C for 5 min). Two percent agarose gel was used to analyze the PCR product of ARMS-PCR. A 100 bp ladder was also loaded alongside the PCR products for comparison of size. The results were analyzed by Wealtec dolphin-doc gel analysis systems.

Statistical analysis
Microsoft office 2016 Excel (Rehmond, WA, USA) was used for organization of the genotyping results. Statistical significance of results was tested using GraphPad Prism software ver8.0.1 (GraphPad Software Inc., San Diego, CA, USA). Genotype distribution of gene polymorphism and their associated strength (odds ratio, OR and relative risk, RR) in patients and healthy control was calculated by two-way Fisher's exact test. Koopman asymptotic score and Baptista-Pike method was computed for the calculation of OR and RR, respectively. A probability of less than 0.05 was taken as significant.

HOPE structural alteration analysis for CTLA-4
HOPE analysis was employed to predict the potential influence of this SNP on protein structure and function. HOPE predicted that CTLA-4 SNP (Variant ID: rs231775) mutation (threonine converted to alanine) leads to substitution of a small sized amino acid residue that is more hydrophobic than the wild type residue. The substitution of alanine may affect the interaction of CTLA-4 with other proteins, and increased hydrophobicity may alter folding of the protein due to loss of H-bonds. The altered amino acid was not located in the conserved region, suggesting that this mutation may not be highly damaging.

CTLA-4 variant influence on therapeutic efficiency of chemotherapeutic drugs
FDA approved Sorafenib is a first line treatment drug, whereas Avastin is a second line treatment drug for HCC. Both drugs were docked through CB-Dock with wildtype and mutated CTLA-4 protein to analyse the influence of SNP on binding energy and interaction of CTLA-4 with Avastin and Sorafenib. Table 2 (Fig. 1a). CTLA-4 + 49A/G polymorphism reduced the number of hydrogen bonds and introduced a covalent bond (Met3) (Fig. 1b). Similar influence of CTLA-4 polymorphism was found for the CTLA-4 and Sorafenib model complex. Sorafenib interacted with CTLA-4 by making two hydrogen bonds (Thr82 and Val84) and ten hydrophobic interactions (Cys85, Glu83, Ala86, Ser101, Asp100, Ile102, Asp123, Leu119, Tyr127 and Arg75). Allele G substitution in CTLA-4 resulted in the addition of four hydrophobic interactions along with a covalent bond ( Fig. 1c and 1d).

In silico prediction of SNP pathogenic impact on IL-4
RegulomeDB ranked the IL-4 SNP at 4, indicating transcription factor binding sites and DNase peak, and a score of 0.60906 suggested high regulatory function of the SNP (Table 1). Alibaba 2.0 transcription site analysis estimated the generation of two additional transcription binding sites for MIG1 and SP1 due to IL-4 rs2243250. SP1 is located within the E1 region that is responsible for enhancer activity. This SP1 site-generating mutation is thought to have no effect on normal functioning of the E1 region, but more research is needed to draw any conclusion [32].

Viral load of HCV in HCV-induced HCC patients
Viral load of HCV was determined through qRT-PCR analysis in the plasma of HCV-induced HCC patients to ensure the HCV infection. Viral load more than 800,000 IU/L is considered high. In this study, high viral load of HCV was observed and mean viral load along

Comparison of CTLA-4 + 49A/G and IL-4 -590 C/T genotypes in HCV-associated HCC patients
Association of CTLA-4 and IL-4 polymorphism with pathogenicity of HCV-induced HCC was evaluated through ARMS-PCR in 213 patient samples. Outcomes indicated that the prevalence of genotype AG (62.9%) in CTLA-4 was more than GG genotype (37.1%) ( Table 3). The OR and RR scores indicated that individuals with AG genotype are more at risk of developing HCV-induced HCC (< 0.0001). IL-4 genotypes CC and CT had association with HCV-induced HCC (< 0.0001).
However, values obtained of RR and OR indicated CC as risk factor genotype and CT as protective genotype for HCV-mediated HCC (Table 3).

Gender-based comparison of CTLA-4 + 49A/G and IL-4 -590 C/T genotypes in HCV-associated HCC patients
Gender based analysis of CTLA-4 + 49A/G and IL-4 -590 C/T genotypes revealed both AG and GG genotypes of CTLA-4 were significantly associated with the disease. AG genotype in both genders was found as pathogenic while GG genotype was indicated as protective (Table 4). Similarly, genotypes CC and CT have significant association with HCV-induced HCC in both females and males. CC genotype in both genders eas indicated as risk factor while CT was shown to be protective ( Table 4).

Association of co-existence of CTLA-4 + 49A/G and IL-4 -590 C/T genotypes in HCV-associated HCC patients
In the present study, the influence of the co-existence of CTLA-4 and IL-4 genotypes in HCV-mediated HCC patients was also evaluated. Genotypes GG (CTLA-4) and TC (IL-4) co-existed in 11.74% of patients and demonstrated significant association with HCV-mediated HCC. However, OR and RR indicated their co-existence as protective. Likewise, genotypes CA (CTLA-4) and TT (IL-4) were found to have co-existence in 8.45% patients with significant association with disease. Contrary to genotypes GG (CTLA-4) and TC (IL-4), genotypes CA (CTLA-4) and TT (IL-4) were determined as risk factor. Similarly, genotypes GA (CTLA-4) and CC (IL-4) were also identified as risk factor and co-existed in around 45% of the patients (Table 5).

Discussion
HCC incidence is decreasing in several high-risk countries, but its incidence is increasing in low-risk countries [33,34]. There are many modified risk factors linked with HCC that increase association of an individual with HCC, such as alcohol intake, smoking or toxin exposure. Apart from extrinsic factors, genetic polymorphism is also identified as a major contributor. HCV infection is among the prominent causes of HCC [35]. The present study aimed to identify whether CTLA-4 + 49 A/G and IL-4-590 C > T polymorphisms associate in HCVinduced HCC Pakistani patients. A further aim was to evaluate potential pathogenic contributions of these polymorphisms to HCC. The IL-4 and CTLA-4 SNPs were localized to the promoter region and protein-coding region, respectively. RegulomeDB analysis indicated likely regulatory roles   [36], Behcet's disease [37], Graves' disease [38], diabetes mellitus, thyroid diseases and colorectal cancer [39]. However, information is lacking on the relationship of the CTLA-4 + 49 A/G polymorphism and HCV induced HCC in the Pakistani population. In a study conducted among Chinese population, CTLA-4 + 49 A/G association with HBV induced HCC was concluded [40]. CTLA-4 polymorphism rs3087243 G > A also showed association with HBV induced HCC among eastern Chinese Han population [17]. CTLA-4 polymorphism along with IL-10 and TNF-alpha also reported associated with HCC [23]. In the present study, CTLA-4 + 49 A/G polymorphism was identified as a potential risk factor for HCV induced HCC in Pakistan. Genotype AG was found to be in high frequency in HCV induced HCC patients while the dominant genotype in control samples was GG. The role of genotype GG was found as protective in HCV-induced HCC patient.
Allele G in CTLA-4 + 49 causes threonine replacement with alanine. Molecular docking of CTLA-4 wildtype and mutated protein with Sorafenib and Avastin indicated that alanine substitution caused an additional covalent bond between CTLA-4 and the drugs. Formation of a covalent bond between ligand and protein is irreversible and a very strong interaction [41]. In most natural biological systems non-covalent interactions are typical [42], and these interactions in comparison to covalent bonding are weak and reversible [41,42]. The presence of a covalent bond between mutated CTLA-4 protein and Avastin and Sorafenib dictates a strong interaction. This further hints towards the protective influence of CTLA-4 GG genotype that might enhance therapy influence.
Studies conducted earlier provided insight into the possible role of IL-4 polymorphism with HCV-induced HCC. The outcomes of the current study pointed towards the association of -590 C > T mutation of IL-4 with increased risk of HCV-induced HCC, and align with a previous study including IL-4 [43]. Annotation scores from RegulomeDB for -590 SNP indicated the regulatory role of this SNP and the presence of transcription factor binding sites. This finding was further confirmed by Alibaba 2.0 analysis. The C > T mutation created 2 additional binding sites, MIG1 and SP1. SP1 is located within the E1 region that is responsible for activity of enhancers in IL-4. Such SP1 mutation was thought to have no effect on normal functioning of the E1 region, but more research is needed to draw any conclusion [44]. These mutations can alter the normal cellular functions, which may lead to cancer formation. These findings support -590 C > T (rs2243250) involvement in hepatocellular carcinoma.
IL-4 -590 C > T polymorphism has a direct association with cancers [24], and is linked with certain diseases such as atopic asthma & allergic rhinitis [25] and smoking linked cancer [36]; however, many studies report that this polymorphism does not have a significant association with disease incidence [45]. A study linked with the Egyptian population showed that IL-4 -590 C > T polymorphism was associated with HCV induced HCC. IL-4 can promote macrophage and Th cell production, which in turn supports progression of cancers like HCC [43]. IL-4 -590 C > T polymorphism is associated with both HBV and HCV induced HCC in a Caucasian population [46]. In our study cohort, genotype "CC" was found to be in high frequency in HCV induced HCC patients and is indicated as risk factor allele.

Conclusion
In the present study, CTLA-4 + 49 genotype AG and IL-4 -590 genotype CC were found to be associated with the pathogenicity of HCV-mediated HCC. Agebased and gender-based analysis revealed the association of CTLA-4 AG genotype with adults (age 20-39) and both genders. Similarly, IL-4 association with age groups 20-39yrs and 40-59yrs and with both genders was revealed. Computation analysis estimated that CTLA-4 polymorphism might have influence on the hydrophobicity and structure of the protein while IL-4 polymorphism might lead to altered transcription factor binding sites in its promoter region. The CTLA-4 polymorphism might augment efficacy of clinically available chemotherapy drugs. Thus CTLA-4 & IL-4 SNP polymorphisms have strong association with HCV induced HCC among Pakistani patients that may have application to serve as genetic markers for preand early diagnosis and prognosis of HCC in HCV patients.